Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin

Vibrio cholerae secretes a large virulence-associated multifunctional autoprocessing RTX toxin (MARTX_Vc). Autoprocessing of this toxin by an embedded cysteine protease domain (CPD) is essential for this toxin to induce actin depolymerization in a broad range of cell types. A homologous CPD is also present in the large clostridial toxin TcdB and recent studies showed that inositol hexakisphosphate (Ins(1,2,3,4,5,6)P₆ or InsP₆) stimulated the autoprocessing of TcdB dependent upon the CPD (Egerer, M., Giesemann, T., Jank, T., Satchell, K. J., and Aktories, K. (2007) J. Biol. Chem. 282, 25314-25321). In this work, the autoprocessing activity of the CPD within MARTX_Vc is similarly found to be inducible by InsP₆. The CPD is shown to bind InsP₆ (K_d, 0.6 μM), and InsP ₆ is shown to stimulate intramolecular autoprocessing at both physiological concentrations and as low as 0.01 μM. Processed CPD did not bind InsP₆ indicating that, subsequent to cleavage, the activated CPD may shift to an inactive conformation. To further pursue the mechanism of autoprocessing, conserved residues among 24 identified CPDs were mutagenized. In addition to cysteine and histidine residues that form the catalytic site, 2 lysine residues essential for InsP₆ binding and 5 lysine and arginine residues resulting in loss of activity at low InsP₆ concentrations were identified. Overall, our data support a model in which basic residues located across the CPD structure form an InsP₆ binding pocket and that the binding of InsP₆ stimulates processing by altering the CPD to an activated conformation. After processing, InsP₆ is shown to be recycled, while the cleaved CPD becomes incapable of further binding of InsP₆.